As part of a continuing study of the neurophysin-peptide hormone system, we are probing the hormone binding site of BNP II in order to determine the array of protein amino acids contributions to the noncovalent binding of ligand. The principle technique that has been used is photoaffinity labelling of bovine neurophysin II (BNP II) with the photolabile tripeptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide. The BNP II protein photolabelled in the hormone binding site was analyzed by peptide mapping and amino acid analysis to determine the site of covalent attachment of the photoaffinity tripeptide. The usefulness of the photoaffinity labelling technique for studying the hormone binding site of neurophysin proteins in general was demonstrated by reacting the photoaffinity tripeptide with ENP I and a fractionated human NP. Additional experiments with photoaffinity labelling could involve further chemical characterization of the reaction between the photoactivated tripeptide and the neurophysin proteins.